Warming Exacerbates the Impact of Nutrient Enrichment on Microbial Functional

1 Warming exacerbates the impact of nutrient enrichment on microbial functional

2 potentials important to the nutrient cycling in shallow lake mesocosms

4 Lijuan Ren 1, Yuanyuan Liu 1, Torben L Lauridsen 2, 3, Martin Søndergaard 2, 3,

5 Boping Han 1, Jianjun Wang 4, Erik Jeppesen 2, 3, 5, 6*, Jizhong Zhou 7, 8,9, and

6 Qinglong L. Wu 2, 4, *

8 1 Department of Ecology and Institute of Hydrobiology, Jinan University, Guangzhou,

9 China

10 2 Sino-Danish Centre for Education and Research, University of Chinese Academy of

11 Sciences, Beijing, China

12 3 Department of Bioscience, Aarhus University, Silkeborg, Denmark

13 4 State Key Laboratory of Lake Science and Environment, Nanjing Institute of

14 Geography and Limnology, Chinese Academy of Sciences, Nanjing, China

15 5 Limnology Laboratory, Department of Biological Sciences and Centre for

16 Ecosystem Research and Implementation, Middle East Technical University, Ankara,

17 Turkey

18 6 Institute of Marine Sciences, Middle East Technical University, Mersin, Turkey

19 7 Institute for Environmental Genomics and Department of Microbiology and Plant

20 Biology, School of Civil Engineering and Environmental Sciences, University of

21 Oklahoma, Norman, OK, USA

22 8 Earth and Environmental Sciences, Lawrence Berkeley National Laboratory,

23 Berkeley, CA, USA

24 9 State Key Joint Laboratory of Environment Simulation and Pollution Control,

25 School of Environment, Tsinghua University, Beijing, China

26 Corresponding authors: Qinglong L. Wu, Email: [email protected]; or Erik

27 Jeppesen, Email: [email protected].

29 Keywords: warming, eutrophication, shallow lake, microbial functional potentials,

30 nutrient cycling

32 Running title: Microbial functional response to warming

33 Table S1 Full names of the abbreviated enzymes/proteins/genes in the main text.

Gene Subcategory Name/abbreviation Enzyme/protein category of the enzyme/protein/gene Carbon Starch amyA Alpha-amylase degradation Starch amyX Pullulanase Starch apu Amylopullulanase Starch cda Cyclomaltodextrinase Starch glucoamylase Glucoamylase Starch pulA Pullulanase Cellulose cellobiase Cellulose Cellulose endoglucanase Endoglucanase Cellulose exoglucanase Exoglucanase Hemicellulose ara Arabinofuranosidase Hemicellulose mannanase Beta-mannanase Hemicellulose xylA Xylose isomerase Hemicellulose xylanase Xylanase Chitin acetylglucosaminidase Acetylglucosaminidase Chitin chitinase Chitinase Chitin endochitinase Endochitinase Chitin exochitinase Exochitinase Pectin pectinase Pectinase Aromatics AssA Alkylsuccinate synthase Aromatics camDCAB Camphor 5-monooxygenase Aromatics limEH Limonene-1,2-epoxide hydrolase Aromatics LMO limonene monooxygenase Aromatics vanA Vanillate demethylase Aromatics vdh Aryl-aldehyde oxidase Lignin phenol_oxidase Phenol oxidase Carbon Reductive aclB ATP citrate lyase fixation tricarboxylic acid cycle Reductive acetyl–CoA CODH Carbon monoxide pathway dehydrogenase Wood-Ljungdahl FTHFS Tetrahydrofolate formylase pathway Calvin cycle rubisco RuBisCo Methane Methanogenesis mcrA Methyl coenzyme M reductase Methane oxidation mmoX Soluable methane monooxygenase Methane oxidation pmoA Particulate methane monooxygenase Nitrogen Ammonification gdh Glutamate dehydrogenase Ammonification ureC Urease Anammox hzo Hydrazine oxidoreductase Assimilatory N nasA Assimilatory nitrate reductase reduction catalytic subunit Assimilatory N nirA Nitrite reductase (NAD(P)H) reduction Assimilatory N nirB Nitrite reductase (NAD(P)H) reduction Denitrification narG Respiratory nitrate reductase alpha chain Denitrification nirS Cytochrome cd1 nitrite reductas 3

Denitrification norB Nitric oxide reductase Denitrification nosZ Nitrous oxide reductase Denitrification nirK Copper containing nitrite reductase Dissimilatory N napA Periplasmic nitrate reductase reduction Dissimilatory N nrfA Ammonia-forming cytochrome reduction c nitrite reductase Nitrification hao Hydroxylamine oxidoreductase Nitrification amoA Ammonia monooxygenase Nitrogen fixation nifH Nitrogenase iron protein Phosphorus Phosphorus utilization phytase Phytase Phosphorus utilization ppk Polyphosphate kinase Phosphorus utilization ppx Exopolyphosphatase Sulfur Adenylylsulfate APS_AprA Adenylylsulfate reductase reductase Adenylylsulfate APS_AprB Adenylylsulfate reductase reductase Sulfide oxidation fccAB Flavocytochrome c sulfide dehydrogenase Sulfide oxidation sqr Sulfide-quinone oxidoreductase Sulfite reductase CysJ Sulfite reductase (NADPH) flavoprotein subunit alpha Sulfite reductase dsrA Dissimilatory sulfite reductase Sulfite reductase dsrB Dissimilatory sulfite reductase Sulfur oxidation sox Sulfer oxidation cycle enzymes

34 Table S2 Permutational multivariate analysis of variance (PERMANOVA) based on

35 Jaccard/Bray-Curtis dissimilarity of overall microbial functional gene structure in

36 different warming and nutrient-enriched treatments. *: p < 0.05; **: p < 0.01.

Jaccard dissimilarity Bray-Curtis dissimilarity Index R2 F p R2 F p Overall warming 0.117 2.251 0.035* 0.125 2.585 0.022* Overall nutrients 0.116 2.286 0.024* 0.127 2.627 0.025*

Overall nutrients: 0.151 2.943 0.013* 0.168 3.466 0.01** overall warming Residual 0.616 0.58

37 Table S3 Permutational multivariate analysis of variance (PERMANOVA) of

38 microbial functional gene structure of the subcategory of C, N, P, and S cycling based

39 on Jaccard/Bray-Curtis dissimilarity. *: p < 0.05; **: p < 0.01.

Bray-Curtis Jaccard dissimilarity Groups dissimilarity F p F p

C Whole 3.084 0.001** 3.621 0.001**

Control vs EW 1.381 0.274 0.364 0.243

Control vs NE 1.575 0.123 1.516 0.116

Control vs NE and EW 4.535 0.025* 3.772 0.028*

EW vs NE 1.708 0.032* 1.650 0.027*

EW vs NE and EW 5.685 0.026* 4.676 0.034*

NE vs NE and EW 5.996 0.03* 4.954 0.029*

Whole 3.581 0.001** 3.581 0.001**

Control vs EW 1.355 0.290 1.348 0.315

Control vs NE 1.479 0.126 1.429 0.113

Control vs NE and EW 4.551 0.026* 3.774 0.031*

EW vs NE 1.619 0.028* 1.575 0.029*

EW vs NE and EW 5.577 0.030* 4.548 0.027*

NE vs NE and EW 6.206 0.038* 5.048 0.032*

Whole 3.442 0.001** 2.975 0.001**

Control vs EW 1.264 0.369 1.270 0.372

Control vs NE 1.403 0.200 1.376 0.127

Control vs NE and EW 4.564 0.031* 3.825 0.031*

EW vs NE 1.469 0.084* 1.447 0.085*

EW vs NE and EW 5.348 0.029* 4.473 0.03*

NE vs NE and EW 5.819 0.023* 4.904 0.031*

Whole 3.707 0.001** 3.158 0.001**

Control vs EW 1.527 0.208 1.493 0.181

Control vs NE 1.614 0.093 1.549 0.088

Control vs NE and EW 4.641 0.029* 3.859 0.026*

EW vs NE 1.672 0.027* 1.627 0.032*

EW vs NE and EW 6.080 0.039* 4.963 0.023*

NE vs NE and EW 6.287 0.024* 5.136 0.036* 40

41 Table S4 Permutational multivariate analysis of variance (PERMANOVA) of

42 microbial functional gene structure composed of the 9619 genes under warming and

43 nutrient treatments based on Jaccard/Bray-Curtis dissimilarity. *: p < 0.05; **: p <

44 0.01.

Jaccard Bray-Curtis

Groups dissimilarity dissimilarity F p F p Whole 6.544 0.001** 9.507 0.001**

Control vs EW 2.491 0.027* 2.295 0.026* Control vs NE 2.495 0.024* 2.265 0.030* Control vs NE and EW 16.197 0.027* 10.459 0.032* EW vs NE 2.077 0.028* 1.947 0.027* EW vs NE and EW 16.941 0.028* 11.154 0.029* NE vs NE and EW 18.549 0.025* 11.943 0.024*

45 Table S5 Water environmental properties in response to warming and nutrient

46 enrichment in the shallow-lake mesocosms. The treatments are termed as follows:

47 ambient temperature, unenriched (control); enhanced warming (A2 + 50%),

48 unenriched (EW); ambient temperature, nutrient-enriched (NE); and enhanced

49 warming (A2 + 50%), nutrient-enriched (NE and EW).

Index Control EW NE NE and EW

Turbidity 6.53±0.98a 5.25±0.18a 11.63±7.84a 15.33±5.41a

3- -1 a ac b ac PO4 -P (mg l ) 0.003±0.0003 0.003±0.0006 0.008±0.001 0.005±0.001

- -1 a a b b NOx -N (mg l ) 0.071±0.066 0.0045±0.0006 3.560±0.218 2.290±0.689

DOC (mg l-1) 1.52±0.11a 1.44±0.06a 2.42±0.41ab 2.76±0.42b

TOC (mg l-1) 1.97±0.30a 1.64±0.08a 4.11±1.77ab 7.68±2.36b

Chlorophyll a (µg l-1) 8.35±4.21a 4.77±1.43a 12.68±2.82a 340.48±153.15b

a EW: Control 0.6 0.4 * 0.2 * 0.0 -0.2

Difference in relative relative in Difference (%) intensity signal i ii iii iv -0.4 b NE: Control 0.6 * 0.4 * 0.2 0.0 * -0.2

Difference in relative in Difference (%) intensity signal i ii iii iv -0.4 c NE and EW: Control 0.6 * * 0.4 0.2 * * 0.0 *

-0.2

Difference in relative in Difference (%) intensity signal i ii iii iv -0.4

Anammox Autotrophy Acetogenesis Nitrification Carbon cycling Denitrification Sulfur cycling MethanogenesisNitrogenNitrogen cyclingAmmonification fixation Carbon degradationMethane oxidation Phosphorus cycling

AssimilatoryDissimilatory N reduction N reduction 50

51 Figure S1 Differences in the microbial metabolic potentials (characterized as the

52 relative signal intensity) involved in the biogeochemical cycling processes of carbon

53 (i), nitrogen (ii), phosphorus (iii), and sulfur (iv). The treatments are termed as

54 follows: ambient temperature, unenriched (control); enhanced warming (A2 + 50%),

55 unenriched (EW); ambient temperature, nutrient-enriched (NE); and enhanced

56 warming (A2 + 50%), nutrient-enriched (NE and EW). EW: Control (a), NE: Control

57 (b), as well as NE and EW: Control (c) means the metabolic potential differences of

58 EW with control, NE with control, NE and EW with control, respectively. *: p < 0.05.

60 Figure S2 Rubisco genes detected in the shallow lake mesocosms. Phylogenetic tree

61 based on the overall rubisco large-subunit amino acid sequences obtained by GeoChip

62 hybridization. Tree topography and evolutionary distance are given by a

63 neighbor-joining method (a); percentages of different types of rubisco genes (b).

64 Phylogenetic tree based on the rubisco large-subunit amino acid sequences, which

65 differed significantly between the warming and nutrient-enriched treatments (c).

a b 0.4 0.4 C N 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0

DCA2 DCA2 -0.1 -0.1 -0.2 -0.2 -0.3 -0.3 -0.4 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 DCA1 DCA1 c d 0.8 0.5 P 0.4 S 0.6 0.3 0.4 0.2 0.2 0.1

DCA2 DCA2 0.0 0.0 Control -0.1 EW -0.2 -0.2 NE NE and EW -0.4 -0.3 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8

66 DCA1 DCA1

67 Figure S3 Microbial functional gene structure of the subcategory of C (a), N (b), P

68 (c), and S (d) cycling in response to warming and nutrients as identified by detrended

69 correspondence analysis (DCA). The treatments are termed as follows: ambient

70 temperature, unenriched (control); enhanced warming (A2 + 50%), unenriched (EW);

71 ambient temperature, nutrient-enriched (NE); and enhanced warming (A2 + 50%),

72 nutrient-enriched (NE and EW).

a b 0.65 0.50 Jaccard’s dissimilarity Bray-Curtis’s dissimilarity 0.60 0.45

0.55 0.40

0.50 0.35

0.45 0.30

Structure dissimilarity Structure Structure dissimilarity Structure 0.40 0.25

0.35 0.20

EW: ControlNE: Control EW: ControlNE: Control NE and EW: Control NE and EW: Control 73

74 Figure S4 Structural dissimilarity of microbial functional genes composing the 9619

75 genes between the different warming and nutrient-enriched treatments and the

76 controls (a: based on Jaccard dissimilarity; b: based on Bray-Curtis dissimilarity). The

77 treatments are termed as follows: ambient temperature, unenriched (control);

78 enhanced warming (A2 + 50%), unenriched (EW); ambient temperature,

79 nutrient-enriched (NE); and enhanced warming (A2 + 50%), nutrient-enriched (NE

80 and EW). EW: Control, NE: Control, and NE and EW: Control mean the structure

81 dissimilarity of EW with control, NE with control, NE and EW with control,

82 respectively.

84 Figure S5 Spearman’s correlations (R values) between transformed environmental and ecosystem metabolic variables and microbial functional

85 gene structure (Bray-Curtis distance) as determined via Mantel tests (*: p < 0.1; **: p < 0.05).

ER 1 DO

Chla 0

CCA2 9.23% CCA2 -1 Control EW NE T -2 NE and EW

-2 -1 0 1 2 3 CCA1 11.7% 86

87 Figure S6 Canonical correlation analysis (CCA) of microbial functional gene

88 structure with environmental/ecosystem metabolic factors in the different warming

89 and nutrient-enriched treatments. Shown are factors significantly explaining the

90 microbial functional gene structure (p < 0.05). The treatments are termed as follows:

91 ambient temperature, unenriched (control); enhanced warming (A2 + 50%),

92 unenriched (EW); ambient temperature, nutrient-enriched (NE); and enhanced

93 warming (A2 + 50%), nutrient-enriched (NE and EW).

2.5

b ) 2.0

-1

-1 1.5

cells l cells

8 1.0

BPR (×10 0.5 a a a

0.0 Control EW NE NE and EW 94

95 Figure S7 Bacterial production rates in the warming and nutrient-enriched treatments.

96 The treatments are termed as follows: ambient temperature, unenriched (control);

97 enhanced warming (A2 + 50%), unenriched (EW); ambient temperature,

98 nutrient-enriched (NE); and enhanced warming (A2 + 50%), nutrient-enriched (NE

99 and EW). Significant (p < 0.05) differences among treatments are indicated by

100 alphabetic letters above the bars according to post hoc comparisons.